Clostridium botulinum is an anaerobic spore-forming bacterium that produces botulinum neurotoxin, the most potent protein toxin, which is known to cause botulism in humans and animals. There are seven serotypes of botulinum neurotoxins (A - G), produced from different strains of C. botulinum, that are physiologically and phylogenetically distinct. The neurotoxin gene cluster, containing the genes for the neurotoxin and the toxin associated proteins, is located on different parts of the genome in different C. botulinum strains, indicating that the gene cluster may be mobile. The overall goal of the PI's research is to understand the biology of the neurotoxin complex and the roles of these neurotoxins in the physiology and evolution of C. botulinum and related species. The study proposed here takes advantage of the recently available C. botulinum type A DNA microarray from NIH/NIAID sponsored Pathogen Functional Genomics Resource Center. We will use these microarray chips to examine, at the whole genome level, the role of neurotoxin complex production and the physiology and evolution of C. botulinum. In Specific Aim 1, we will perform comparative genomic microarray analyses to characterize the genomes of various C. botulinum type A strains and compare them to strains that did not acquire the toxin gene cluster. Genes unique to the toxin-producing strains will be identified and further characterized. In Specific Aim 2, we will determine the growth phase-dependent gene expression profiling of C. botulinum in response to the botulinum neurotoxin production levels. Using the microarray analyses, we will examine and compare the global gene transcriptional profiles between toxin-producing wild-type strains and toxin-deficient mutant strains. The impacts of the neurotoxin on cellular functions, growth and sporulation will be analyzed. The knowledge gained in the proposed studies will help us understand the biology of toxin production in C. botulinum with the intent of contributing to the body of knowledge about the neurotoxin and developing novel strategies to prevent botulism. PUBLIC HEALTH RELEVANCE: Clostridium botulinum produces botulinum neurotoxin, which is the most potent protein toxin known to cause botulism in humans and animals. Due to the potency of the toxin, the bacterium and its neurotoxins are classified as the "Category A" Select Agents for their potential use as biological weapons. The proposed studies allow further understanding of the biology of toxin production in C. botulinum with the intent of contributing to the body of knowledge about the neurotoxin and developing novel strategies to prevent botulism.